Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised.

Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.

Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos.

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos.

The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers.

Acute decreases in nutrient intake can improve embryo quality in sheep, although reductions in ovulation rate can also occur. In cattle, short-term nutrient restriction prior to ovulation has been shown to increase subsequent pregnancy rates. Thus, the objective was to determine the effect of a severe reduction in food intake on follicle growth and embryo quantity and quality in heifers superovulated with FSH. Beef heifers (n = 61) were offered a diet of grass silage and concentrates (ratio of 5:1, on a fresh weight basis), which was adjusted to provide a predicted intake of 28.6 Mcal/kg ME/d (H) or 9.6 Mcal/kg ME/d (L). Heifers were synchronized with a progesterone-releasing device for 7 d. They were allocated to oocyte recovery (n = 16/treatment) after 3 (225 IU) or 8 (600 IU) injections of FSH given at 12-h intervals. Oocytes were matured, fertilized and cultured individually in vitro. The remaining heifers (n = 14/treatment) were superovulated using FSH (600 IU), and embryos were recovered 7 d after breeding. The embryos were morphologically graded and subsequently cultured for 24 h before differential staining to determine inner cell mass and trophectoderm cell numbers. Follicle numbers increased following 8 (16.6 +/- 2.0) compared with 3 (6.7 +/- 0.6) injections of FSH (P < 0.0001). Heifers on the L diet had more follicles than those on the H diet (13.5 +/- 2.4 vs 9.6 +/- 1.2; P < 0.06), which was predominantly due to an increase in the number of 7- to 10-mm follicles. However, this effect was only evident after 8 injections of FSH. There was no nutritional effect on cleavage rates in vitro (55.6 +/- 8.1 vs 53.8 +/- 9.0 for H vs L diets, respectively). However, cleavage rates were lower in oocytes collected after 8 than after 3 injections of FSH (31.3 vs 69.2%; P < 0.0001). There was no significant effect of nutrition on ovulation rate after FSH (14.4 +/- 1.9 vs 16.3 +/- 3.0 for H vs L diet, respectively). The number of embryos recovered was not different between heifers on H (10.4 +/- 1.3) and L (11.3 +/- 2.4) diets. Following culture for 24 h, a significantly higher proportion of embryos from heifers on the L diet developed to the blastocyst stage (72.9 vs 41.5%; P < 0.01). Total cell numbers on Day 8 were greater in embryos from heifers on the L diet (98.3 vs 75.4; P < 0.0001); yet the inner cell mass as a percentage of total cells was not different (21 vs 20%). These data indicate that low energy intake prior to and during superovulation resulted in more follicles and in improved embryo quality, as evident from the increased number of blastocysts formed and higher cell numbers.

Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes.

In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca2+ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca2+ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP3), used to test the sensitivity of the InsP3R, released significantly less Ca2+ in calf than in cow oocytes, whereas higher concentrations of InsP3 (500 microM) released maximal [Ca2+]i in both oocytes. These results suggested that the Ca2+ content of intracellular stores was similar, but the sensitivity of the InsP3R may be different. Following insemination, calf oocytes exhibiting [Ca2+]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation.

Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization.

The objective of these experiments was to evaluate factors affecting in vitro fertilization of bovine oocytes matured in vitro, and their subsequent development to blastocysts. In Expts 1 and 2, sperm concentration, spermatozoa and oocyte incubation time, motility enhancers and semen source were manipulated. Fluorescent microscopy of microtubules and chromatin was used to observe sperm penetration rate, sperm aster formation and chromatin decondensation. Oocyte penetration rates were affected by sperm concentration but not by spermatozoa and oocyte incubation time. The effect of sperm concentration was due primarily to changes in polyspermy and not monospermy. Motility enhancers had no effect on any parameter measured. In Expt 3, oocytes were matured for 17, 22, 28 and 34 h before fertilization and evaluated for fertilization rates, morphology of cortical granules and exocytosis and blastocyst development. A domain free of cortical granules that was associated with the metaphase chromatin was not observed in mature bovine oocytes. As oocytes matured from 17 to 34 h, the distribution of cortical granules progressed from clustered to diffuse. Although monospermic fertilization rates were similar and cortical granule exocytosis occurred in all groups, polyspermy increased with maturation time. Development to blastocysts increased from 17 to 22 h of maturation but decreased thereafter with increasing maturation time. These results suggest that polyspermy can be reduced by adjusting sperm concentration and spermatozoa and oocyte incubation time with little effect on monospermic fertilization. Increased polyspermy with increased maturation time was not due to a lack of cortical granule exocytosis.

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes.

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage.

The effects of once or twice daily injections of pFSH on superovulatory response in heifers.

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.

Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs.

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.

In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid.

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.

Comparison of two progestogens during out-of-season breeding in a commercial ewe flock.

A comparison was made of the relative effectiveness of subcutaneous ear implants containing 2 mg Norgestomet or vaginal pessaries containing 60 mg medroxyprogesterone acetate (MAP) to induce estrus and conception in dry anestrous ewes. Groups of ewes were treated with one of the two progestogens for 14 d, and 500 IU pregnant mare serum gonadotropin (PMSG) was administered intramuscularly at the time of progestogen withdrawal. No significant differences in estrus induction, pregnancy rate or number of lambs born per ewe lambing were observed. Ewes treated with Norgestomet had 96% estrus, 60% pregnancy rate and 1.4 lambs per ewe lambing. Comparably, ewes treated with MAP had 94% estrus, 65% pregnancy rate and 1.7 lambs per ewe lambing. Norgestomet implants compared favorably with MAP pessaries for estrus induction and breeding of commercial, dry anestrous ewes.

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin.

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.

In vitro steroidogenesis by granulosa cells from equine pre-ovulatory follicles.

Twenty-three follicles were collected from 14 mares on specific days and grouped to represent follicles from early (Group 1; n = 6), mid (Group 2; n = 11) and late (Group 3; n = 6) oestrus, as described previously (Tucker et al., 1988). Isolated granulosa cells (GC) from each follicle were cultured in multiwell plates containing either Eagle's Minimum Essential Medium (MEM) alone, eLH (300 ng/ml), eFSH (300 ng/ml) or eLH + eFSH (300 ng/ml each), in the presence or absence of 0.5 microM testosterone. Media were collected and replaced at 24 h of culture, and 24 h later, media were again collected and cultures terminated. Media were assayed for oestradiol-17 beta (E2), progesterone (P4), androstenedione (A) and testosterone (T) by radioimmunoassay (RIA). Media containing exogenous T were assayed for E2 and P4. There was no effect of exogenous gonadotrophins on steroid secretion by GC from any group, so these values were pooled and averaged. Steroid concentrations were expressed as ng/ml/1000 live cells seeded (mean +/- s.e.m.). Progesterone was the predominant steroid secreted by GC from all groups without added T after 24 h of culture, and was highest in media from Group 3 (7.86 +/- 1.19 ng/ml) 1000 cells; vs Group 1 (0.02 +/- 0.004 ng/ml/1000 cells) and Group 2 (0.15 +/- 0.03 ng/ml/1000 cells; P less than 0.001). Androstenedione was the predominant androgen secreted, but its levels did not change from group to group. Oestradiol and T levels were comparable in media from Group 1 cultures (E2: 0.003 +/- 0.0004 ng/ml/1000 cells; T: 0.003 +/- 0.0009 ng/ml/1000 cells), but E2 was highest in Group 2 media (0.17 +/- 0.11 ng/ml/1000 cells; P less than 0.001) and T levels were greatest in Group 3 (0.02 +/- 0.003 ng/ml/1000 cells; P less than 0.005). In the presence of exogenous T, E2 increased in media from all groups (P less than 0.0001). Oestradiol levels from Group 1 (0.52 +/- 0.12 ng/ml/1000 cells) and Group 3 (0.57 +/- 0.09 ng/ml/1000 cells) cultures were not different, but were highest in media from Group 2 (2.15 +/- 0.29 ng/ml/1000 cells; P less than 0.01). By 48 h, P4 secretion was higher in all groups (P less than 0.0001) and was the predominant steroid secreted by Group 3 GC. Progesterone levels were not affected by the addition of T. Androstenedione increased in Group 3 media (P less than 0.001), whereas E2 and T remained unchanged. These results suggest that the pattern of equine GC steroid secretion changes as follicular maturation proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)

N-glycosylated and unglycosylated forms of caprine trophoblast protein-1 are secreted by preimplantation goat conceptuses.

Goat conceptuses secrete caprine trophoblast protein-1 (cTP-1) which is related antigenically to the abundant embryonic interferon-alpha II of sheep and cattle. Antiserum to ovine and bovine TP-1s immunoprecipitated three molecular weight classes (23,000, 21,000 and 17,000, each with two isotypes) of cTP-1 from goat conceptus culture medium. Cultures which contained tunicamycin resulted in a shift in the Mr = 23,000 and Mr = 21,000 forms to a Mr of 17,000. The Mr = 23,000 and 21,000 forms, but not the Mr = 17,000 form, bound to Concanaval in A-Sepharose and were eluted under conditions selective for glycoproteins bearing complex-type oligosaccharide(s). Thus cTP-1 is a mixture of glycosylated and unglycosylated polypeptides.

Rabbit sperm function after co-culture with uterine epithelial cells.

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy.

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of passive immunization against estradiol on rabbit ovum transport.

The role played by estradiol in control of ovum transport was studied in rabbits that were induced to ovulate by hCG stimulation. Withholding estrogen from target tissues during ovum transport by passive immunization with sheep anti-estradiol immunoglobulin (AE) resulted in accelerated oviductal transport and expulsion of ova from the uterus. The degree of accelerated transport was dependent on the duration of AE treatment. When AE treatment was started 24 h before hCG, fewer ova were recovered from the reproductive tracts at 72 h after hCG than were recovered from tracts of animals treated with a nonspecific immunoglobulin; the location of ova at 48 h after hCG was unaltered by this AE regimen. When AE treatment was started 72 h before hCG, fewer ova remained in the oviducts than were found in the tubes of controls at 48 h after hCG; and at 48 h after hCG, ova were found in the uteri of animals that had been treated with AE starting 72 h before hCG. When AE treatment was started 48 h before hCG, the position of eggs within the reproductive tract was not different from controls at 48 h after hCG. These observations support the concept that estrogen withdrawal is involved in the transport of ova through the oviduct of the rabbit, and suggest that the lack of estrogen secretion from the time of ovulation through the transport period is important in the control of normal ovum transport.

Progesterone synthesis and histology of postpartum bovine corpora lutea.

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.

Attempts surgically to induce chorionic fusion between embryos.

Intercornual fistulas were made in the uteri of 19 ewes in an attempt to induce chorionic fusion between fetuses. Ewes were 28 to 40 days pregnant with at least one fetus per horn. Sections were removed from the dorsomedial wall of each uterine horn, and left and right horns were sutured together around the openings to produce the fistulas. In some cases a doughnut-shaped sheet of silastic was inserted between the horns, and in most cases the chorioallantoic membranes of the respective fetuses were sutured to one another prior to suturing the uterine walls. In the 16 animals in which intercornual fistulas were constructed in the area of the intercornual ligament, no chorionic anastomosis was induced. In one of the three animals in which fistulas were produced distal to that ligament, the fistula remained patent 25 days post-surgery, and membranes of two female fetuses were joined, through the opening, by a narrow strand of chorionic tissue. We concluded that this technique was not a promising one for induction of choriovascular anastomosis.

Residues in colostrum following antibiotic dry cow therapy.

Cows from five dairy herds were used to determine persistence of antibiotic residues in colostrum and milk following dry cow therapy. Cows were treated in all quarters at drying off with antibiotics approved for use for nonlactating cows. Antibiotics procaine penicillin G plus dihydrostreptomycin, novobiocin, cloxacillin, or cephapirin were compared with no treatment. Composite colostrum samples were collected from each cow at first milking after parturition. Samples were screened for residues by Delvotest P. Colostrum samples positive by Delvotest also were tested by Bacillus stearothermophilus disc assay. Four of 186 colostrum samples from cows treated with antibiotics at drying off were positive for residues by Delvotest. Only one was confirmed positive by disc assay following heat treatment. All colostrum samples from 48 cows not treated were negative. Samples of first marketable milk also were collected. Over 96% of milk samples from cows treated at drying off and 100% of milk samples from cows not treated were negative for residues by Delvotest. If manufacturer's recommendations are followed, antibiotic residues in colostrum and milk following dry cow therapy with products in our study should not be a significant problem.

Residues in young veal calves after consumption of milk containing penicillin.

Residues in urine, kidney, and muscle following consumption of milk containing procaine penicillin G were determined in 4- to 8-day-old Holstein bull calves. Calves were fed milk diluted 1:1 with water. Nine control calves received diluted milk only while 8 calves received diluted milk containing 6,600 IU penicillin/kg (low group), and 10 calves received 13,200 IU/kg of milk consumed (high group). Urine samples were collected over 11 h after each morning feeding for 3 days. Calves were slaughtered on day 4 of trial and kidney, muscle, and urine samples obtained. Urine was tested for residues with the Live Animal Swab Test and tissues by the Swab Test on Premises. Both tests are microbiological assays used for detecting growth inhibition of Bacillus subtilis. No residues were detected in urine of control calves. Residues in urine were detected in 6 calves in the low group and 7 calves in the high group after feeding. At slaughter, residues were in 3 of 5 urine samples from calves in the low group and 8 of 10 calves in the high group. However, no residues were detected in kidney or muscle of control or treated calves.